Abstract

Bioinformatics and mass spectrometry (MS)-based proteomics are specialitiesthat became an integral part of biological research following the human genomesequencing project whereas the area of molecular systems biology (MSB) later emergedas a new approach to decoding life. The practice of MSB has made importantcontributions in our understanding of complex biological processes, primarily throughmodelling of high-throughput gene expression measurements at the level of transcription.Proteomics techniques have been successfully applied for systematic protein identification,characterization of protein-protein interactions and post-translational modifications.Practical large-scale quantitation, however, has traditionally been a considerableanalytical challenge for MS-based proteomics technologies and this has hindered theconstruction of models based on protein expression data. In recent years this picturehas begun to change with the rebirth of multiple reaction monitoring (MRM) MS and theemergence of targeted proteomics, where accurate quantitative expression measurementsare obtained in a rapid and cost-effective manner for pre-specified sets of biologicallyinteresting proteins. We employed triple quadrupole (TQ) instrumentation for measuringthe expression of key proteins involved in the execution and regulation of the inammatoryresponse. We also perturbed the response through activation of neuroendocrine pathwaysdownstream of the corticotropin releasing hormone (CRH) transmembrane receptor(CRH-R), in an effort to reveal novel dynamic changes in protein expression inducedby CRH. The information required for the facilitation of the targeted experiment waspreviously obtained from: i) several large-scale protein identification experiments whichinvolved the utilisation of a recent two-dimensional liquid chromatography fractionationtechnique and ii) de novo MRM assay development based on public datasets. Thetime-series expression data generated were processed and analysed by in-house developedsoftware tools and finally modelled in an attempt to deduce relevant regulatory proteinnetworks. Although the biological importance of CRH, a major component of thephysiological stress system, in the context of local inammation has been highlightedbefore, yet, as in cases of other neuropeptides, its immunoregulatory role remains unclear.The MSB approach employed here has enabled us to suggest potentially importantcomponents of the CRH effect. In the near future, we anticipate that the targetedproteomics approach will be an important ingredient of MSB research.